JP2010512747A - Compositions and methods for treating muscular diseases and cardiovascular disorders - Google Patents

Abstract

  The present invention relates to a novel microRNA, mir-208-2, involved in muscle disease and cardiovascular disorders. The invention also relates to oligonucleotide therapeutic agents (antisense oligonucleotides and / or double stranded oligonucleotides such as dsRNA) and its in the treatment of muscular diseases and cardiovascular disorders caused by dysregulation of mir-208-2. Regarding use.

Description

FIELD OF THE INVENTION This invention relates to a novel microRNA, mir-208-2, involved in muscular diseases and cardiovascular disorders. The invention also relates to oligonucleotide therapeutics (antisense oligonucleotides and / or double stranded oligonucleotides) and their use in the treatment of muscular and cardiovascular disorders caused by dysregulation of mir-208-2.

Background MicroRNA (miRNA) is a group of non-coding RNA genes whose final product is a functional RNA molecule of ˜22 nt. It is processed as a single stranded RNA from an endogenously encoded incomplete hairpin precursor. This is considered to have a function of suppressing translation by base pairing with the 3 ′ untranslated region (UTR) of the target mRNA (Griffith-Jones et al., 2006, Nucleic Acids Research, Vol. 34, D140). -D144).

MicroRNA (miRNA) biosynthesis is a complex, multi-step process. Primary miRNA transcripts are transcribed by RNA polymerase II and can range in size from hundreds to thousands of nucleotides in length (pri-miRNA). miRNAs can be traced back to two genomic sources. Some miRNAs are located in intron regions of protein coding genes. Others are located in introns or exons of non-coding RNA. Interestingly, a pri-miRNA can encode a single miRNA, but may contain a diverse cluster of miRNAs. The pri-miRNA is then processed to a ~ 70 nt hairpin (pre-miRNA) by the nuclear ribonuclease III (RNase III) endonuclease, Drosha. pre-miRNA is excreted from the nucleus to the cytoplasm by Exporin5 / RanGTP. In the cytoplasm, the secondary RNase III, Dicer, along with its dsRBD protein partner, cleaves the pre-miRNA in the stem region of the hairpin, releasing an RNA duplex of ˜21 nucleotides. One strand of the miRNA duplex enters the RNA-induced silencing complex (RISC), which is a protein complex that suppresses the expression of the target gene, and the other strand is degraded. Strand selection depends on the local thermodynamic stability of the miRNA duplex. The strand with the 5 ′ end forming a less stable pair is loaded into the RISC complex. The miRNA loaded on the RISC complex is considered to have a function of suppressing translation by forming a base pair with the 3 ′ untranslated region (UTR) of the target mRNA (Du, T. and Zamore, PD et. al. micro-Primer: the biogenesis and function of microRNA. Development (2005) 132, 4645-4652). Currently, 462 human miRNA sequences have been deposited with miRBase ( http://microrna.sanger.ac.uk) , suggesting that this list will reach 800 points. The identification of a large number of miRNAs so far suggests that they can have a complex role in the control and fine-tuning of biological processes. In fact, various miRNAs regulate cell proliferation (mir-125b and let-7), hematopoietic B cell lineage (mir-181), B cell survival (mir-15a and mir-16-1), brain patterning (Mir-430), pancreatic cell insulin secretion (mir-357), adipocyte development (mir-375), and muscle growth and differentiation (miR-1 and miR-133). Many miRNAs are located in genomic regions involved in cancer. For example, a cluster containing mir-16-1 and mir-15 eliminates and down-regulates much of B-cell chronic lymphocytic leukemia (B-CLL; Calin, GA, et al. MicroRNA-Cancer connection: The beginning of a new tale. Cancer Res. (2006) 66, 7390-7394).

  Effective therapeutic treatment of diseases and disorders that can be caused by dysregulated microRNAs has not been met and is continually needed. The present invention provides compounds that meet this need and provides other advantages. The compounds of the present invention are nucleic acids that can specifically target and treat dysregulated microRNAs. One group is antisense DNA or RNA; the other group is double stranded RNA (dsRNA). These also include a group known as small interfering RNA (siRNA).

  siRNAs are a novel group of therapeutic agents known to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) Discloses the use of dsRNA of at least 25 nucleotides to inhibit gene expression in C. elegans. siRNA can also be used in plants (see, eg, WO 99/53050, Waterhouse et al .; and WO 99/61631, Heifetz et al.), in Drosophila (eg, Yang, D., et al., Curr. Biol. (2000) 10 : 1191-1200) and other organisms, including mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.) Have been shown to degrade target RNA. This natural mechanism has attracted attention to develop a new class of agents for treating disorders caused by genetic abnormalities or unwanted regulation.

  Despite significant advances in the field of RNAi, there remains a need for a variety of drugs that can treat diseases caused by novel molecular pathologies, such as dysregulated microRNAs.

SUMMARY OF THE INVENTION We have identified a new miRNA, mir-208-2, that satisfies the above needs. The present invention therefore relates to an isolated nucleic acid molecule of less than 500 nucleotides, characterized in that it comprises mir-208-2 (SEQ ID NO: 7). In one embodiment, an isolated nucleic acid molecule comprising mir-208-2 (SEQ ID NO: 7) has a length of less than 200 nucleotides, for example to target pri-miRNA. In other embodiments, an isolated nucleic acid molecule comprising mir-208-2 (SEQ ID NO: 7) has a length of less than 80 nucleotides, eg, to target pre-miRNA.

  A specific embodiment of the present invention is an isolated nucleic acid molecule consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or selected from the group consisting of SEQ ID NO: 7.

  One skilled in the art will readily appreciate that the scope of the present invention also includes isolated nucleic acid molecules of less than 500 nucleotides consisting of nucleic acid sequences complementary to those described above. For example, an isolated nucleic acid consisting of SEQ ID NO: 8.

Another aspect of the present invention is an isolated nucleic acid molecule having a length of 8-50 nucleotides, wherein said isolation is under physiological conditions, such as in a cell (cytoplasm or nucleus) or under conditions simulating such conditions. An isolated nucleic acid molecule that can hybridize with a nucleic acid molecule and inhibit the function of mir-208-2 (SEQ ID NO: 7), eg, binding of mir-208-2 to its target.
A specific example of such a molecule is an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 77.

  The isolated nucleic acid molecule has one or more chemical modifications selected from, for example, a) a 3 ′ cap, b) a 5 ′ cap, c) a modified internucleoside linkage, or d) a modified sugar or base moiety. Those skilled in the art will readily understand that they can be supported.

  A nucleic acid vector comprising the nucleic acid and at least one vector propagation sequence is also an aspect of the present invention. The invention also relates to a nucleic acid vector comprising the nucleic acid and at least one vector propagation sequence.

The isolated nucleic acid molecule of the present invention or the nucleic acid vector of the present invention can be used as a medicament, for example, in a lipid or polymer-based pharmaceutical delivery system.
Such a medicament of the present invention can be used to treat a muscle disease in a subject having a muscle disease.
A specific embodiment of the muscle disease is a cardiovascular disorder. Accordingly, a medicament according to the invention for treating a cardiovascular disorder in a subject or for the manufacture of a medicament for the treatment of a muscular disease or cardiovascular disorder.

  The present invention also includes kits for use in diagnosing cardiovascular disorders or determining treatment strategies. Typically, the kit comprises a nucleic acid reagent of the invention in RNA, DNA, mixed RNA or DNA, and optionally a nucleic acid reagent comprising any chemical modification.

The nucleic acid molecules of the invention can also be used for any other purpose, such as for experimental purposes. Accordingly, the present invention includes a method of increasing or decreasing the expression of mir-208-2, wherein the isolated nucleic acid molecule of the present invention is used, for example, in a cell.
Similarly, the nucleic acid molecules of the invention can also be used as diagnostic probes or as experimental probes.

  Furthermore, the present invention has found that two other miRNAs, miRNAs, mir-208 and mir-499 are closely related to the expression of mir-208-2. Accordingly, the present invention is also a use for the manufacture of a medicament for the treatment of a muscular disease or cardiovascular disorder or for the diagnosis of a muscular disease or cardiovascular disorder, comprising mir-208 (SEQ ID NO: 6). An isolated nucleic acid molecule of less than 500 nucleotides, and / or mir-499 (SEQ ID NO: 9), and / or mir-208 (sequence of No. 6) or the use of an isolated nucleic acid molecule of less than 500 nucleotides comprising the complementary sequence of mir-499 (SEQ ID NO: 9).

Alignment of introns of MYH6 with human mir-208 and zebrafish, human, chimpanzee, dog and rat with mir-208-2. MYH6, MYH7 and 18S RT-PCR on RNA isolated from different developmental mouse hearts. Northern blot analysis of mir-208, mir-208-2, mir-499 and mir-206 on RNA isolated from different developmental mouse hearts. U6 snRNA was used as a loading target. Lanes 11-14 correspond to 50 pg synthetic miRNA.

Detailed description of the invention:
The present invention relates to the discovery of a novel microRNA, mir-208-2, involved in muscle disease or cardiovascular disorders. It also relates to oligonucleotide therapeutics (antisense DNA / RNA and / or double stranded RNA) and their use in the treatment of muscular diseases or cardiovascular disorders caused by mir-208-2 dysregulation.

  mir-208-2 is a gene located in intron 30 of the MYH7 gene. MYH7 (GenBank accession number 00257: on chromosome 14). MYH7 is a motor contractile protein consisting of a spherical head (including actin and ATP binding sites) followed by a rod-like tail sequence, and is one of the structural blocks of a thick myosin filament. Each myosin filament contains two heavy chains and four light chains. Myocardial contraction rate is regulated by the degree of ATPase activity in the head region of the myosin molecule. The major determinant of myosin ATPase activity, and thus muscle contraction rate, depends on the relative amounts of the two myosin heavy chain isomers, MYH6 and MYH7. The MYH6 isoform that exhibits high ATPase activity has approximately four times the enzyme activity of MYH7. Both MYH6 and MYH7 are expressed in different amounts in the human heart. In the failing human heart, MYH6 mRNA and protein levels are downregulated, while MYH7 is upregulated.

  The transcription of mir-208-2 is closely related to the MYH7 transcription because it does not have an independent promoter. Therefore, this is transferred only when MYH7 is transferred. It is believed that the mir-208-2 microRNA is released from the MYH7 transcript when the pre-mRNA is processed and the intron is removed. The remaining intron sequence of intron 30 is further processed to produce full length mir-208-2.

  mir-208-2 is highly conserved among vertebrates. FIG. 1 shows the alignment of mir-208-2 between multiple species. Also shown is the similarity of mir-208 and another microRNA present in intron 28 of MYH6.

The mir-208-2 gene identified by the inventors has the following sequence (including flanking regions: the actual mir-208-2 sequence is underlined in bold):

For sequence comparison, mir-208 from intron 28 of known MYH6 is also provided.

機能性ガイド配列およびアンチガイド配列であると結論されるこのマイクロＲＮＡの最も高度に保存されている部分を同定することが可能である。 FIG. 1 shows the remarkable conservation of mir-208-2 among various species. From this alignment, the following:

It is possible to identify the most highly conserved portions of this microRNA that are concluded to be functional guide sequences and anti-guide sequences.

Unlike MYH6 and MYH7, MYH7B (GenBank accession number 02084: on chromosome 20) is not well characterized. Based on the high degree of homology with MYH7, MYH7B is classified as a slow MYH isoform. To date, the clear function of MYH7B has not been described in the literature. Furthermore, there are no disease links that contribute to MYH7B dysfunction. Interestingly, like MYH6 and MYH7, MYH7B also contains a miRNA, mir-499, in one of the introns.

  Bioinformatics analysis and the examples below show that a novel microRNA, mir-208-2, is involved in the regulation of signal transduction pathways involved in cardiac hypertrophy. Thus, this microRNA is extremely useful as a target for the treatment of disorders and diseases that may be associated therewith.

  We disclose herein various methods and compositions that have therapeutic utility. As a general overview, it is believed that reducing the level of target microRNA can provide therapeutic benefit in some cases. Such reduction can be readily obtained by the use of antisense nucleic acid molecules, such as antisense DNA, that bind directly to the target miRNA, such as mir-208-2. In other cases, an increase in the level of the target microRNA, such as mir-208-2, can benefit. Such elevation can be readily obtained through the use of sense nucleic acid molecules and several dsRNA molecules, eg, several miRNAs that bind to the miRNA target and act synergistically with the miRNA. The present invention therefore relates to both types of therapeutic agents.

  The following definitions are used throughout this specification and the claims.

  An “isolated nucleic acid molecule” means that the material is removed from its original environment (eg, its natural environment if it is naturally occurring). For example, a naturally occurring polypeptide present in a living animal has not been isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system is Even if it is reintroduced into Such a polynucleotide may be part of a vector and / or such a polynucleotide may be part of a composition and the vector or composition is not part of its natural environment, so Have been separated.

  A “nucleic acid vector” is a nucleic acid sequence designed to proliferate and / or transcribe when exposed to a cellular environment, such as a cell lysate or whole cell. “Gene therapy vector” means a nucleic acid vector that also carries a functional aspect for transfecting whole cells with the intention of increasing the expression of one or more genes and / or proteins. In each case, such vectors usually contain a “vector propagation sequence” which is a replication origin recognized by the cell, generally allowing the vector to propagate in the cell. A wide range of nucleic acid vectors and gene therapy vectors are well known to those skilled in the art.

  As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to therapeutic benefits in the treatment, prevention or management of pathological conditions mediated by dysregulation of mir-208-2. Means the amount to provide. The specific amount that is therapeutically effective is readily determined by the ordinary health care professional, and includes, for example, the type of morbidity, the history and age of the patient, the stage of the morbidity and the combined administration of other drugs Can vary depending on factors known in the art.

  As used herein, a “pharmaceutical composition” includes a pharmacologically effective amount of a therapeutic agent of the present invention and a pharmaceutically acceptable carrier. As used herein, a “pharmacologically effective amount”, “therapeutically effective amount” or simply “effective amount” is used to refer to an agent effective to obtain the intended pharmacological, therapeutic or prophylactic result. Means quantity. For example, if a given clinical treatment is considered effective when there is at least a 25% decrease in a measurable parameter associated with the disease or disorder, the therapeutically effective amount of the agent for treatment of the disease or disorder is This is the amount necessary to reduce the parameter by at least 25%.

  The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term explicitly excludes cell culture media. Pharmaceutically acceptable carriers for oral administration of the drug are pharmaceutically such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents and preservatives. Including but not limited to acceptable excipients. Suitable inert diluents include sodium and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, with corn starch and alginic acid being the preferred disintegrants. Binders can include starch and gelatin, and when present, the lubricant is generally magnesium stearate, stearic acid or talc. If desired, tablets can be coated with materials such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

  As used herein, the expression “muscle disease” includes, but is not limited to, heart disease. This expression relates to any type of degenerative muscle disease where the primary pathology may be a decrease in striated muscle weight and / or a decrease in its function. This includes, but is not limited to, muscular dystrophy, trauma and myasthenia gravis.

  As used herein, a “transformed cell” is a cell into which a vector capable of expressing a dsRNA molecule has been introduced. Cells containing exogenously supplied nucleic acids, such as transiently or stably transfected agents of the present invention, are also considered transformed cells.

  Primary miRNA transcripts are transcribed by RNA polymerase II and can range in size from hundreds to thousands of nucleotides in length (pri-miRNA). A pri-miRNA can encode a single miRNA, but may include a variety of miRNA clusters. The pri-miRNA is then processed to a ~ 70 nt hairpin (pre-miRNA) by the nuclear ribonuclease III (RNase III) endonuclease, Drosha. Accordingly, the isolated nucleic acid molecules of the present invention have a variety of preferred lengths based on the intended target. When targeting pri-miRNA, preferred lengths of about 500 nucleotides, eg, 499, 450, 400, 350, 300, 250 nucleotides can be used. When targeting pre-miRNA, the preferred length varies from 100 to 200 nucleotides in length, such as 100, 120, 150, 180 or 200 nucleotides in length. In the cytoplasm, the secondary RNase III, Dicer, along with its dsRBD protein partner, cleaves the pre-miRNA in the stem region of the hairpin, releasing an RNA duplex of ˜21 nucleotides. Thus, for example, 80, 70, 60, 50, 40, 30, 25, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8 nucleotides in length isolated polynucleotide Is also considered an aspect of the present invention.

  The inventors have discovered that an inhibitor of mir-208-2 is injected into a fertilized egg of a zebrafish (Dario rerio) leading to a dramatic decrease in cardiac function (blood circulation, heart rate, etc.). “Function of mir-208-2 (SEQ ID NO: 7)” can therefore be assessed in this assay or a similar assay. Another possibility for assaying “function of mir-208-2 (SEQ ID NO: 7)” is the interaction of mir-208-2 with its target, eg binding to it.

  In practicing the present invention, many conventional techniques in molecular biology, microbiology and recombinant DNA are used. These techniques are well known, for example, Current Protocols in Molecular Biology, Volumes I, II, and III, 1997 (FM Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (DN Glover ed.); Oligonucleotide Synthesis, 1984 (ML Gait ed.); Nucleic Acid Hybridization, 1985, (Hames Transcription and Translation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986 (RI Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, A Practical Guide to Molecular Cloning the series, Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells, 1987 (JH Miller and MP Calos eds., Cold Spring Harbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 ( Wu and Grossman, and Wu, eds., Respectively).

Therapeutic Agents Certain therapeutic agents of the invention are described herein as siRNAs comprising mir-208-2 anti-guide and guide sequences for use in increasing or decreasing mir-208-2 activity in a cell. . Another therapeutic agent of the invention is an antisense DNA or RNA composition useful for reducing mir-208-2 activity in cells.

dsRNA Pharmaceuticals The siRNA molecules of the present invention mediate RNA interference ("RNAi"). The term “RNAi” is well known in the art and is generally understood to mean the inhibition of one or more target genes in a cell by siRNA having a region complementary to the target gene. A variety of assays are known in the art to test for the ability of dsRNA to mediate RNAi (see, eg, Elbashir et al., Methods 26 (2002), 199-213). The effect of the dsRNA of the invention on gene expression is typically at least 10%, 33%, 50%, 90%, 95% of the target gene when compared to cells not treated with the RNA molecule of the invention. Or 99% inhibition. The “siRNA” or “small interfering ribonucleic acid” of the present invention has a known meaning in the art including the following aspects. siRNAs consist of two strands of ribonucleotides that hybridize with complementary regions under physiological conditions. The strands are usually separated. Because the two strands have distinct roles in the cell, one strand is referred to as the “antisense” strand, also known as the “guide” sequence, which is due to cleavage in the functional RISC complex. Used to guide it to the correct mRNA. Because of its association with RNA compounds, this use of “antisense” is different from the antisense DNA compounds described elsewhere herein. The other strand is known as the “antiguide” sequence, which is known as the sense strand because it contains the same nucleotide sequence as the target sequence. The chains can be joined by molecular linkers in certain embodiments. Individual ribonucleotides can be unmodified, naturally occurring ribonucleotides, unmodified, naturally occurring deoxyribonucleotides, or they can be chemically modified as described elsewhere herein. Or may be synthetic.

  As used herein, a dsRNA contains two basic groups. The siRNA exists. However, when the two strands are part of one larger molecule, therefore, there is a nucleotide break between the 3 ′ end of one strand and the 5 ′ end of the other strand forming each duplex structure. When joined by a non-strand, the bound RNA strand is referred to as a “hairpin loop”, “short hairpin RNA” or “shRNA”. shRNA is usually transcribed from a nucleic acid vector and expressed in the target cell of interest.

The nucleic acid molecule of the invention may be any dsRNA comprising SEQ ID NO: 7 or SEQ ID NO: 8, and the same cellular mRNA, such as mir-208-2, and / or mir-208 as defined in the claims. -2 Targets itself.

  The nucleic acid molecule of the present invention comprises a region substantially identical to a region of the target gene mRNA. A region that is 100% identical to the corresponding sequence of the target gene is preferred. This state is referred to as “fully complementary”. However, in view of the nature of miRNA and its mechanism of action, the region may also be 1, 2 or 3 compared to the corresponding region of the target gene, depending on the length of the targeted mRNA region. It may contain mismatches, or more, and may not itself be completely complementary. However, the most important feature is that the molecule can specifically bind mir-208-2 under physiological conditions, for example in cells. In certain embodiments, the RNA molecules of the present invention specifically target one predetermined gene. In order to target only the desired mRNA, the siRNA agent has 100% homology with the target mRNA and has at least two mismatched nucleotides with all other genes present in the cell or organism. Methods for analyzing or identifying siRNAs with sufficient sequence identity to effectively inhibit the expression of specific target sequences are known in the art, for example, the method described in WO2005 / 059132. Sequence identity can be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991 and references cited therein), and the percent difference between nucleotide sequences. Can be calculated using standard parameters, eg, by the Smith-Waterman algorithm as implemented in the BESTFIT software program (eg, University of Wisconsin Genetic Computing Group).

  The region length of the siRNA complementary to the target of the present invention can be 10-100 nucleotides, 12-25 nucleotides, 14-22 nucleotides or 15, 16, 17 or 18 nucleotides. When there is a mismatch to the corresponding target region, the length of the complementary region generally needs to be somewhat longer.

  Because each siRNA can carry overhang ends (which may or may not be complementary to the target) or additional nucleotides that are complementary to itself but not to the target gene, each individual siRNA The total length of the strand can be 10-100 nucleotides, 15-49 nucleotides, 17-30 nucleotides or 19-25 nucleotides.

  The phrase “each strand is less than 49 nucleotides” includes all modified and unmodified nucleotides but does not contain any chemical moieties that may be added to the 3 ′ or 5 ′ end of the strand. Means the total number. Although the short chemical moieties inserted into the strand are not counted, a chemical linker designed to join two separate strands is not considered to create a contiguous nucleotide.

  The phrase “at least one 1-6 nucleotide overhang at the 5 ′ end or 3 ′ end” refers to the structure of a complementary siRNA that forms from two separate strands under physiological conditions. When the terminal nucleotide is part of the double stranded region of the siRNA, the siRNA is considered to have a blunt end. When one or more nucleotides are not paired at the end, an overhang occurs. Overhang length is measured by the number of overhanging nucleotides. Overhanging nucleotides can be present at the 5 'or 3' end of either strand.

The siRNA of the invention exhibits high in vivo stability by including at least one modified nucleotide in at least one strand and may be particularly suitable for oral delivery. Accordingly, the siRNA of the invention comprises at least one modified or non-natural ribonucleotide. A lengthy description of many known chemical modifications is given in PCT published patent application WO 200370918 and will not be repeated here. Suitable modifications for delivery are:
a) 3 'cap;
b) 5 'cap;
c) modified internucleoside linkages; or d) chemical modifications selected from modified sugar or base moieties.

  Suitable modifications are sugar moieties (ie 2′-position of the sugar moiety, eg 2′-O- (2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), ie, alkoxyalkoxy groups) or base moieties (ie, non-natural or modified bases that maintain the ability to pair with other specific bases of another nucleotide chain). Other modifications include, but are not limited to, so-called “backbone” modifications, including substitution of phosphate ester groups (bonding of adjacent ribonucleotides with, for example, phosphorothioate, chiral phosphorothioate or phosphorodithioate).

  Terminal modifications, also referred to herein as 3 'caps or 5' caps, can be important. The cap may consist of simply adding additional nucleotides such as “TT” known to confer stability to the siRNA. The cap may consist of a more complex chemistry known to those skilled in the art.

〔ＸはＯまたはＳであり、
Ｒ１およびＲ２は独立してＯＨ、ＮＨ２、ＳＨ、アルキル、アリール、アルキル−アリール、アリール−アルキルであり、ここでアルキル、アリール、アルキル−アリール、アリール−アルキルはさらなるヘテロ原子および官能基、好ましくはＮ、ＯもしくはＳから選択されるヘテロ原子またはＯＨ、ＮＨ２、ＳＨ、カルボン酸もしくはエステル基から選択される官能基で置換されていてもよい；
またはＲ１およびＲ２は式Ｙ−Ｚであってよく、ここでＹはＯ、Ｎ、Ｓであり、ＺはＨ、アルキル、アリール、アルキル−アリール、アリール−アルキルであり、ここでアルキル、アリール、アルキル−アリール、アリール−アルキルはさらなるヘテロ原子、好ましくはＮ、ＯまたはＳの群から選択されるヘテロ原子で置換されていてもよい〕
の化合物から選択される。 In one embodiment, the 3 ′ cap is a chemical group attached to the 3 ′ end via the 3 ′ carbon and has the formula I

[X is O or S;
R1 and R2 are independently OH, NH2, SH, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl are further heteroatoms and functional groups, preferably Optionally substituted with a heteroatom selected from N, O or S or a functional group selected from OH, NH2, SH, carboxylic acid or ester groups;
Or R1 and R2 may be of the formula YZ, where Y is O, N, S and Z is H, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, Alkyl-aryl, aryl-alkyl may be substituted with further heteroatoms, preferably heteroatoms selected from the group of N, O or S]
Selected from the following compounds:

  Examples of modifications for the sugar moiety include 2 'alkoxy ribonucleotides, 2' alkoxy alkoxy ribonucleotides, locked nucleic acid ribonucleotides (LNA), 2'-fluoro ribonucleotides, morpholino nucleotides.

  Internucleoside linkages can also be modified. Examples of internucleoside linkages include phosphorothioate, phosphorodithioate, phosphoramidate and amide linkages.

  R1 can be OH.

  R1 and R2 may contain 1 to 24 C atoms, 1 to 12 C atoms, 2 to 10 C atoms, 1 to 8 or 2 to 6 C atoms in total. Good. In other embodiments, R1 and R2 are independently OH, lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl, wherein lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl May be substituted with further heteroatoms and functional groups as defined above. In other embodiments, both R1 and R2 are not OH.

  The term “lower” refers to a compound or group that, in relation to an organic group or compound, can be branched or unbranched containing no more than 7 carbon atoms, preferably 1-4 carbon atoms. To do. Lower alkyl means, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and branched pentyl, n-hexyl and branched hexyl.

  Examples of alkoxy include O-Met, O-Eth, O-prop, O-but, O-pent, O-hex.

  Methods for synthesizing siRNA containing at least one modified or non-natural ribonucleotide are well known to those skilled in the art and are readily available. For example, various synthetic chemistry are described in PCT published patent publications WO2005021749 and WO200370918, both of which are hereby incorporated by reference. The reaction is carried out in solution or preferably on a solid phase, or with a polymer-supported reactant, after which the synthetic RNA strand is mixed under conditions that form siRNA capable of intervening RNAi. Can do.

  The present invention also includes siRNA comprising at least one modified nucleotide suitable for oral delivery. In a functional sense, this means that the siRNA has suitable pharmacokinetics and biodistribution to achieve delivery to the intended target tissue by oral administration. Among other things, this requires serum stability, lack of immune response, and drug-like kinetics. Many of these features of siRNA can be revealed based on standard gastric acid assays and standard serum assays described elsewhere herein.

  Although specific therapeutic agents can be designed in a variety of forms, certain functional features distinguish preferred dsRNA from other dsRNAs. Among other things, features such as good serum stability, high potency, lack of inducible immune response, and good drug-like kinetics (all measurable by those skilled in the art) to identify preferred dsRNAs of the invention To be tested. In some situations, all of these functional aspects are not present in the preferred dsRNA. However, one skilled in the art can optimize these variables and others for selecting preferred compounds of the invention.

  Any method can be used to administer the dsRNA of the invention to a mammal containing mir-208-2 dysregulated. For example, administration can be local (eg, vaginal, transdermal, etc.); orally; or parenterally (eg, subcutaneous, intraventricular, intramuscular, or intraperitoneal injection, or intravenous infusion). Administration can be rapid (eg, by injection) or can occur over an extended period of time (eg, by slow infusion or a delayed release formulation).

  For example, dsRNA formulated with or without liposomes can be applied topically directly to the tissue of interest. For topical administration, dsRNA molecules can be formulated into formulations such as sterile and non-sterile aqueous solutions, common solvents such as non-aqueous solutions of alcohol, or liquid or solid oil-based solutions. Such a solution may also contain buffers, diluents and other suitable additives. Topical compositions can be formulated in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Gels and creams can be formulated using polymers and penetrants known in the art.

  For parenteral, intrathecal or intraventricular administration, dsRNA molecules can be formulated into compositions such as sterile aqueous solution which may also contain buffers, diluents and other suitable additives ( For example, permeation enhancers, carrier compounds and other pharmaceutically acceptable carriers).

  In addition, dsRNA molecules can be administered to mammals using non-viral methods such as the biological or non-biological methods described in, for example, US Pat. No. 6,271,359. Non-biological delivery includes (1) loading a liposome with a dsRNA acid molecule provided herein; (2) mixing the dsRNA molecule with a lipid or liposome to form a nucleic acid lipid or nucleic acid liposome complex. Or (3) can be accomplished by a variety of methods including, but not limited to, obtaining a polymer-based therapeutic delivery system. These techniques are generally well known in the art. A brief description follows.

  Liposomes can consist of cationic and neutral lipids commonly used to transfect cells in vitro. Cationic lipids can be complexed with negatively charged nucleic acids (eg, via charge) to form liposomes. Examples of cationic liposomes include, but are not limited to, lipofectin, lipofectamine, lipofectase and DOTAP. Methods for forming liposomes are well known in the art. Liposome compositions are formed, for example, from phosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylglycerol or dioleoylphosphatidylethanolamine. A variety of lipophilic reactants are commercially available, including Lipofectin.RTM. (Invitrogen / Life Technologies, Carlsbad, Calif.) And Effectene.TM. (Qiagen, Valencia, Calif.). In addition, systemic delivery methods can be optimized using commercially available cationic lipids such as DDAB or DOTAP that can be mixed with neutral fats such as DOPE or cholesterol, respectively. In some cases, liposomes such as those described in Templeton et al. (Nature Biotechnology, 15: 647-652 (1997)) can be used. In other embodiments, polycations such as polyethyleneimine can be used to deliver in vivo and in vivo (Boletta et al., J. Am Soc. Nephrol. 7: 1728 (1996)). For further information on the use of liposomes to deliver nucleic acids, see US Pat. No. 6,271,359, PCT Publication WO 96/40964 and Morrissey, D. et al. 2005. Nat Biotechnol. 23 (8): 1002-7 Can do.

  Biological delivery can be accomplished in a variety of ways, including but not limited to the use of viral vectors. For example, viral vectors (eg, adenovirus and herpes virus vectors) can be used to deliver shRNA molecules to skin cells and cervical cells. Using standard molecular biology techniques, one or more of the shRNAs provided herein can be introduced into one of the viral vectors that have been developed to deliver nucleic acids to cells. These resulting viral vectors can be used to deliver one or more dsRNAs to cells, for example by infection.

  The dsRNA of the invention can be formulated in a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier” (also referred to herein as an “excipient”) is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle. It is. Pharmaceutically acceptable carriers can be liquid or solid and can be selected to achieve bulkiness, homogeneity, and other suitable transportability and chemical properties in view of the intended mode of administration. . Typical pharmaceutically acceptable carriers are, for example, water; saline; binders (eg, polyvinylpyrrolidone or hydroxypropylmethylcellulose); bulking agents (eg, lactose and other sugars, gelatin or calcium sulfate); Including, but not limited to, excipients (eg starch, polyethylene glycol or sodium acetate); disintegrants (eg starch or starch glycolate sodium); and wetting agents (eg sodium lauryl sulfate).

  In addition, the dsRNA of the invention can be formulated into a composition that is mixed, encapsulated, or complexed or otherwise bound to a mixture of other molecules, molecular structures or nucleic acids. For example, a composition comprising one or more dsRNA agents of the invention can be combined with other therapeutic agents used to treat similar disorders.

  Cell culture or laboratory animal standards to measure the toxic and therapeutic effects of such compounds, for example LD50 (dose at which 50% of the population is lethal) and ED50 (dose at which 50% of the population is therapeutically effective) Can be measured by conventional pharmaceutical methods. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred.

  Data from cell culture assays and animal studies are used in dosage formulations for use in humans. The dosage of the composition of the invention is generally in the range of circulating concentrations including ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration utilized. For compounds used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Dosages are circulating plasma concentration ranges that include the IC50 (ie, the concentration of the test compound that results in half-maximal inhibition of symptoms) of the compound or, where appropriate, the polypeptide product of the target sequence in an animal model, as measured in cell culture. Can be formulated (eg, to reduce the concentration of the polypeptide). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography. In any case, the administering physician may adjust the amount and timing of dsRNA administration based on results known using the standard effect measurement methods known in the art or as described herein. it can.

Antisense DNA Therapeutic Agents The antisense oligonucleotides of the present invention (sometimes referred to herein as “antisense”) are designed to target mir-208-2 and reduce its transcriptional level. The antisense itself may target any part of this mir-208-2 to knock down its intracellular level.

  Antisense compounds are commonly used as research and diagnostic agents and have been the subject of therapeutic development for many years. Antisense oligonucleotides can inhibit gene expression with excellent specificity and are often used by those skilled in the art to elucidate the function of a particular gene. Antisense compounds are also used, for example, to abolish the function of members of various biological pathways.

  Antisense specificity and sensitivity are also utilized by those skilled in the art for therapeutic use. Antisense oligonucleotides have been used as therapeutic tools in the treatment of animal and human disease states. Antisense oligonucleotides are safe and effective for human administration, and many clinical trials are still in progress. Thus, it has been established that oligonucleotides can be useful therapeutic methods that can make treatment regimes useful for the treatment of cells, tissues and animals, especially humans. In the context of the present invention, the term “antisense” refers to an oligomer or polymer of deoxyribonucleic acid (DNA), or a mimetic thereof. The term includes oligonucleotides consisting of oligonucleotides having naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages, and non-naturally occurring moieties with similar functions. Such modified or substituted antisense is often preferred over the native form because of desirable properties such as, for example, enhanced cellular uptake, improved nucleic acid target affinity, and improved stability in the presence of nucleases.

  Although antisense oligonucleotides are the preferred form of antisense compounds, the present invention includes other oligomeric antisense compounds, including but not limited to oligonucleotide mimics such as: The antisense compounds of the present invention preferably contain about 8-30 nucleobases. Particularly preferred are antisense oligonucleotides comprising about 8-30 nucleobases (ie, about 8-30 linked nucleosides). As is known in the art, a nucleoside is a combination of base and sugar. The base portion of the nucleoside is usually a heterocyclic base. The two most common groups of such heterocyclic bases are the purine group and the pyrimidine group. A nucleotide is a nucleoside that further comprises a phosphate group covalently linked to the sugar portion of the nucleoside. In nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2 ', 3' or 5 'hydroxyl group of the sugar. In the formation of oligonucleotides, phosphate groups are covalently bonded to adjacent nucleoside groups to form linear polymeric compounds. Each end of this linear polymer-like structure can in turn be further joined to form a ring structure, but a ring-opened linear structure is generally preferred. In the oligonucleotide structure, the phosphate group is generally said to form the internucleoside backbone of the oligonucleotide. The normal linkage or DNA backbone is a 3 'to 5' phosphodiester linkage.

  Specific examples of preferred antisense compounds useful in the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined herein, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not retain a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified oligonucleotides that do not carry a phosphorus atom in the internucleoside backbone are also considered to be oligonucleosides.

  Preferred modified oligonucleotide backbones are, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates and chiral phosphonates, phosphonates, phosphinates Phosphoramidates including 3'-aminophosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphotriesters, and boranos with conventional 3'-5 'linkages Phosphate, these 2'-5 'linked analogs, 3'-5' to 5'-3 'or 2'-5' to 5'-2 'linked adjacent pairs of nucleoside units having opposite polarity Including what is. Various salts, salt mixtures and free acid forms are also included. Techniques for synthesizing antisense compounds comprising oligonucleotides having the modified backbone or non-natural internucleoside linkage can be performed using conventional methodologies and are familiar to those skilled in the art. Representative US patents that teach the production of such phosphorus-containing linkages include, but are not limited to, US Pat. Nos. 3,687,808; 4,469,863 and 5,625050, each of which is hereby incorporated by reference in its entirety. As part of).

  Preferred modified oligonucleotide backbones that do not contain a phosphorus atom are short chain alkyl or cycloalkyl internucleoside linkages, mixtures of heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatoms or heterocyclic nucleosides. It has a skeleton formed by bonding. These are morpholino linkages (partially formed from the sugar portion of the nucleoside); siloxane backbone; sulfide, sulfoxide and sulfone backbone; formacetyl and thioform acetyl backbone; methylene form acetyl and thioform acetyl backbone; alkene Including skeletons; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and others with mixed N, O, S and CH 2 component moieties. Such oligonucleotides can be synthesized by those skilled in the art by conventional methods, such as those described in US Pat. Nos. 5,034,506; 5,166,315 or 5,677,439, each of which is herein incorporated by reference in its entirety. Some).

  In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, ie the backbone of the nucleotide unit, are replaced with new groups. The base unit is maintained for hybridization with the appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have outstanding hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of the oligonucleotide is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobase is retained and linked directly or indirectly to the aza nitrogen atom of the backbone amide moiety. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, US Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference in its entirety. And). Further teachings of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

  The most preferred embodiment of the present invention is an oligonucleotide having a morpholino backbone structure described in US Pat. No. 5,034,506. Also preferred are oligonucleotides having a phosphorothioate backbone and heteroatom backbones, especially —CH 2 —NH—O—CH 2 —, —CH 2 —N (CH 3) —O—CH 2-[methylene (described in US Pat. No. 5,489,677). Known as methylimino) or MMI skeleton], —CH 2 —O—N (CH 3) —CH 2 —, —CH 2 —N (CH 3) —N (CH 3) —CH 2 — and —O—N (CH 3) —CH 2 —CH 2. -Wherein the natural phosphodiester backbone is represented by -OPPO-CH2- and oligonucleosides having an amide backbone as described in US Pat. No. 5,602,240.

  The modified oligonucleotide may also have one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2 'position: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; Or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted with C1-C10 alkyl or C2-C10 alkenyl and alkynyl or unsubstituted. Particularly preferred are O [(CH2) nO] mCH3, O (CH2) nOCH3, O (CH2) nNH2, O (CH2) nCH3, O (CH2) nONH2, and O (CH2) n ON [(CH2) nCH3)] 2 (where n and m are 1 to about 10). Other preferred oligonucleotides comprise one of the following at the 2 ′ position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, Chemical groups that improve the pharmacokinetic properties of the oligonucleotide, or chemical groups that improve the pharmacodynamic properties of the oligonucleotide, and other substituents that have similar properties. A preferred modification is 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O- (2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta 1995, 78, 486-504), ie alkoxyalkoxy groups. Further preferred modifications include 2'-dimethylaminooxyethoxy, i.e. O (CH2) 2ON (CH3) 2 group (also known as 2'-DMAOE). A further preferred modification of this category is a modification of the bicyclic group known collectively as LNA (locked nucleic acids) as described in Rajwanshi et al., Angew. Chem. Int. Ed. 2000, 39, 1656-1659. It is.

  Other preferred modifications include 2'-methoxy (2'-O-CH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications may be made at other positions on the oligonucleotide, particularly at the 3 'position of the sugar of the 3' terminal nucleotide or 2'-5 'linked oligonucleotide and at the 5' position of 5 'terminal nucleotide. . Oligonucleotides may have sugar mimetics such as cyclobutyl moieties instead of pentofuranosyl sugars. One skilled in the art can produce such modified sugar structures using routine methods. Representative US patents that teach the manufacture of such modified sugar structures include, but are not limited to, US Pat. Nos. 4,981,957; 5,118,800 and 5,700,920, each of which is incorporated herein by reference in its entirety. Part).

  Antisense oligonucleotides may also contain nucleobases (often referred to in the art simply as “bases”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil. (U) is included. Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and guanine 6-methyl and Other alkyl derivatives, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine , 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo 5-trifluoromethyl and Other 5-substituted uracils and cytosines, including 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaguanine, and 3- deazaguanine and 3-deazaadenine. Additional nucleobases are described in U.S. Pat.No. 3,687,808, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B. ed., CRC Press, 1993 Including Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include N-2, N-6 and O-6 substituted purines, 5-propyluracil and 5-propynylcytosine, including 5-substituted pyrimidines, 6-azapyrimidines and 2-aminopropyladenine. 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2 ° C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), which is a further particularly preferred base substitution when combined with a 2′-O-methoxyethyl sugar modification.

  One skilled in the art can produce modified nucleobases by methods well known in the art. For example, representative U.S. patents that teach the production of the above modified nucleobases and other modified nucleobases include U.S. Pat.No. 3,687,808, and U.S. Pat.Nos. 4,845,205; 5,130,302 and 5,134,066. Without limitation (each of which is incorporated herein by reference in its entirety).

  Other modifications of the oligonucleotides of the invention are chemically linked to one or more moieties or conjugates of the oligonucleotide that improve activity, cellular distribution or cellular uptake of the oligonucleotide. Such groups include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let , 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), fatty chains such as dodecanediol or undecyl residues (Saison -Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54) Phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et a l., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotide, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229 -237), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Representative US patents that teach the manufacture of such oligonucleotide conjugates include, but are not limited to, US Pat. Nos. 4,828,979; 4,948,882 and 5,688,941, each of which is herein incorporated by reference in its entirety. Some).

  Not all positions of a given compound need to be uniformly modified, and in fact, multiple such modifications may also be incorporated in a single compound or a single nucleoside within an oligonucleotide. The invention also includes antisense compounds that are chimeric compounds. A “chimeric” antisense compound or “chimera” in the context of the present invention is an antisense compound, in particular two or more made up of at least one monomer unit, ie a nucleoside in the case of oligonucleotide compounds. An oligonucleotide having a chemically apparent region. These oligonucleotides are typically modified at least to impart oligonucleotides with increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid, Includes one region. The additional region of the oligonucleotide functions as an enzyme substrate capable of cleaving RNA: DNA or RNA: RNA hybrids. As an example, RNAase H is a cellular endonuclease that degrades the RNA strand of an RNA: DNA duplex. Thus, activation of RNase H causes cleavage of the RNA target, thereby greatly improving the efficiency of oligonucleotide inhibition of gene expression. As a result, similar results can often be obtained with shorter oligonucleotides when using chimeric oligonucleotides compared to phosphorothioate deoxyoligonucleotides that hybridize to the same target region.

  The chimeric antisense compounds of the invention can be formed as a composite structure of two or more oligonucleotides, modified oligonucleotides, oligonucleotides and / or oligonucleotide mimics described above. Such compounds are also referred to in the art as hybrids or gapmers. One skilled in the art can produce the hybrid structure by conventional methods. Representative US patents that teach the manufacture of such hybrid structures include, but are not limited to, US Pat. Nos. 5,013,830; 5,149,797 and 5,700,922, each of which is hereby incorporated by reference in its entirety. And).

  The antisense compound used in the present invention can be produced simply and very commonly by a well-known solid phase synthesis technique. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art can additionally or alternatively be used. It is well known to use similar techniques to produce oligonucleotides such as phosphorothioates and alkylated derivatives.

  Furthermore, those skilled in the art will readily appreciate that the antisense compounds of the present invention do not target mir-208-2 itself, and may target mRNA containing mir-208-2, such as pri-miRNA or pre-miRNA. You will understand.

  Table 1 shows preferred antisense sequences that down-regulate mir-208-2. These sequences may be used with any chemical modification disclosed herein.

  The antisense compounds of the invention are synthesized in vitro, but may include antisense compositions of biological origin, or gene vector constructs designed to participate in the in vivo synthesis of antisense molecules. The compounds of the present invention may also be mixed, encapsulated or otherwise used to assist in uptake, distribution, and / or absorption, eg, as liposomes, receptor targeting molecules, oral, rectal, topical or other formulations. It may be complexed or otherwise bound with a molecule, molecular structure or mixture of molecules, and conventional methods for doing so exist and are well known to those skilled in the art. For example, representative US patents include, but are not limited to, US Pat. Nos. 5,108,921; 5,354,844 and 5,595,756, each of which is hereby incorporated by reference in its entirety.

  The antisense compounds of the invention can be any pharmaceutically acceptable salt, ester or salt of such ester, or a biologically active metabolite when administered (directly or indirectly) to an animal, including a human. Or any other compound from which that residue can be obtained. Thus, for example, this disclosure also describes prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents. The term “pharmaceutically acceptable salt” refers to a physiologically and pharmaceutically acceptable salt of a compound of the invention: ie, retains the desired biological activity of the parent compound and confers undesirable toxicity to it. It means salt without any problems. Such compounds can be prepared by one skilled in the art according to conventional methods (Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66, 1-19).

  The term “prodrug” refers to a drug manufactured in an inactive form that is converted into the active form (ie, drug) in the body or cells by the action of endogenous enzymes or other chemicals and / or conditions. In particular, prodrug versions of the oligonucleotides of the invention can be prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives as WO 93/24510 (Gosselin et al., Published 9 December 1993) or WO 94 / 26764 (Imbach et al.).

  The antisense compounds of the present invention can also be used for diagnosis, treatment, prevention and as research kits. For therapy, an animal suspected of having a disease or disorder that can be treated by modulating the expression of mir-208-2, preferably a human, is treated by administering an antisense compound of the invention. The compounds of the present invention can be used as pharmaceutical compositions by adding an effective amount of an antisense compound and a suitable pharmaceutically acceptable diluent or carrier. The use of antisense compounds and the methods of the invention are also prophylactically useful, for example for the prevention or delay of infection, inflammation or tumor formation.

  The antisense compounds of the present invention hybridize with nucleic acids encoding mir-208-2 and allow easy construction of sandwich and other assays that take advantage of this fact for research and diagnostics. Useful for. Hybridization of the antisense oligonucleotide of the present invention and a nucleic acid encoding mir-208-2 can be detected by methods known in the art. Such methods may include enzyme-oligonucleotide conjugation, oligonucleotide radiolabeling or any other suitable detection method. Kits using such detection methods for detecting the level of mir-208-2 in a sample can also be produced.

  The invention also includes pharmaceutical compositions and formulations comprising the antisense compounds of the invention. The pharmaceutical compositions of the invention can be administered in a variety of ways depending on whether local or systemic treatment is desired or on the area to be treated. Administration is topical (mucosa including eye and vaginal and rectal delivery), lung, eg by inhalation or insufflation of powder or aerosol contained in nebulizer; intratracheal, intranasal, epithelial and transdermal), oral or parenteral possible. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracerebral, eg, intrathecal or intraventricular. Oligonucleotides with 2'-O-methoxyethyl modifications are believed to be particularly useful for oral administration.

Use of the Compositions of the Invention As noted above, the compositions of the present invention find use in multiple embodiments, including but not limited to research, diagnosis and treatment.

  For therapeutic use, the compositions described herein can be used to treat conditions caused by disease and dysregulation of mir-208-2. As described herein, this novel microRNA was found to be expressed primarily in muscle tissue, particularly heart tissue, and was found to be associated with MYH7 transcription and expression. Diseases associated with dysregulation of mir-208-2 include, but are not limited to, muscular diseases and heart disorders, and may include such diseases associated with mutations in MYH6, MYH7, or MYH7B. The dysregulated expression of miRNA may be responsible for disease progression and is therefore qualified as a potential therapeutic target by inhibition of miRNA or reintroduction of dsRNA using siRNA or shRNA and a suitable delivery system.

  For example, the compounds of the present invention can be used to treat the most common persistent arrhythmia, atrial fibrillation (AF), which is extremely structural, contractile for the purpose of stabilizing AF for long periods of time. Is related to sex and electrophysiological remodeling (Allessie, M., J. Ausma, and U. Schotten, Electrical, contractile and structural remodeling during atrial fibrillation. Cardiovasc Res, 2002. 54 (2): p 230-46). AF is associated with increased expression of intraventricular myosin isoforms in atrial muscle and is considered part of the differentiation process. Interestingly, it was found that in AF myocardium, the functional class of genes characteristic of ventricular muscle is up-regulated, while the functional class mainly expressed in atrial muscle is down-regulated. (Barth, AS, et al., Reprogramming of the Human Atrial Transcriptome in Permanent Atrial Fibrillation: Expression of a Ventricular-Like Genomic Signature 10.1161 / 01.RES.0000165480.82737.33. Circ Res, 2005. 96 (9): p. 1022-1029). One of the genes found to be up-regulated in AF is MYH7B, a gene that contains mir-499 in one of the introns. The isozyme shift of MYH family members observed in human atrial tissue is thought to be an initial adaptation to hemodynamic overload (Buttrick, PM, et al., Myosin isoenzyme distribution in overloaded human atrial tissue. Circulation, 1986. 74 (3): p. 477-83; Yazaki, Y., et al., Molecular adaptation to pressure overload in human and rat hearts. J Mol Cell Cardiol, 1989. 21 Suppl 5: p. 91-101. ).

  In other examples, the compounds are used in hypertrophic cardiomyopathy (HCM) characterized by small, markedly hypertrophic hypercontracted left ventricle (LV) (Maron, BJ, Hypertrophic cardiomyopathy: a systematic review. Jama, 2002. 287 (10): p. 1308-20). Human ventricular muscle expresses both MYH6 and MYH7, with MYH7 being predominant. There is no significant difference in MYH7 expression even during hypertrophy. However, it is believed that overloaded human ventricular muscle loses the small amount of MYH6 normally contained because this form is not detected in autopsy material of patients with hypertensive disease or in intraoperative biopsy of patients with valve heart disease. (Schwartz, K., et al., Left ventricular isomyosins in normal and hypertrophied rat and human hearts.Eur Heart J, 1984. 5 Suppl F: p. 77-83, Mercadier, JJ, et al., Myosin isoenzymes in normal and Hypertrophied human ventricular myocardium. Circ Res, 1983. 53 (1): p. 52-62).

  The identified microRNAs, mir-208-2, mir-208 and mir-499 can also be used as biomarkers in the detection of premature atrial fibrillation or hypertrophic cardiomyopathy.

  Pharmaceutical compositions or formulations for topical administration of a compound of this invention include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves, etc. are also useful.

  Compositions and formulations for oral administration include powders, granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavors, diluents, emulsifiers, dispersants or binders may be desired.

  Compositions or formulations for parenteral, intrathecal or intraventricular administration include sterile aqueous solutions, which also include buffers, diluents and other suitable additives, such as permeation enhancers, simplex compounds and other pharmaceutical agents. May include, but is not limited to, an acceptable carrier or excipient.

  The pharmaceutical compositions of the present invention include solutions, emulsions, and liposome-containing formulations. These compositions can be prepared by those skilled in the art according to conventional methods from a variety of ingredients including, but not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

  The pharmaceutical composition of the present invention, which may be conveniently present in unit dosage form, can be prepared by conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredient with a pharmaceutical carrier or diluent. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or well-dispersed solid carriers or both, and then if necessary shaping the product.

  The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

  In one embodiment of the invention, the pharmaceutical composition can be formulated and used as a foam. Pharmaceutical foams include, but are not limited to, formulations such as emulsions, microemulsions, creams, jelly and liposomes. Although basically the same properties, these formulations differ in the composition and concentration of the final product. Methods for preparing such compositions and formulations are generally known to those skilled in the pharmaceutical arts and can be applied to formulating the compositions of the present invention.

  The composition can be administered alone or in combination with at least one other agent, such as a stabilizing compound, which includes, but is not limited to, saline, buffered saline, dextrose and water. It can be administered in a sterile, biologically compatible pharmaceutical carrier. The composition can be administered to the patient alone or in combination with other drugs, medicaments or hormonal agents.

  The pharmaceutical composition included in the present invention can be applied to various routes such as oral, intravenous, intramuscular, intraarticular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, Administration can be by, but not limited to, enteral, topical, sublingual or rectal means. In addition to the active ingredient, these pharmaceutical compositions comprise a suitable pharmaceutically acceptable carrier containing excipients and auxiliaries that facilitate the processing of the active compound into a pharmaceutically usable formulation. May be. More detailed techniques for formulation and administration can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.). The pharmaceutical composition for oral administration can be formulated at a dosage suitable for oral administration using a pharmaceutically acceptable carrier well known to those skilled in the art. Such carriers make it possible to formulate pharmaceutical compositions into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for inoculation to patients. For oral pharmaceutical preparations, the active ingredient and solid excipients are mixed, the mixture obtained is ground if desired, the appropriate auxiliaries are added, and the mixture of granules is processed to obtain a tablet or dragee core be able to. Suitable excipients are carbohydrate or protein extenders such as sugars such as lactose, sucrose, mannitol or sorbitol; corn, wheat, rice, potato or other plant derived starches; cellulose such as methylcellulose, hydroxypropylmethyl- Cellulose or sodium carboxymethylcellulose; gums such as gum arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof such as sodium alginate.

  Dragee cores are used in combination with concentrated coatings, including suitable coatings such as gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. obtain. Coloring agents or coloring agents can also be added to the tablets or dragee coatings to characterize the product identification and amount of active compound, eg, dosage.

  Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules may contain active ingredients mixed with a bulking or binding agent such as lactose or starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquids, or liquid polyethylene glycol, with or without stabilizers.

  Pharmaceutical compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. In addition, suspensions of the active compounds can be formulated as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers can also be used for delivery. If desired, the suspension may contain suitable stabilizers or agents that improve the solubility of the compound to enable the production of highly concentrated solutions.

  For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

  The pharmaceutical compositions of the present invention can be manufactured by methods known in the art, for example, by conventional mixing, dissolving, granulating, sugar-coating, grinding, emulsifying, capsule filling, filling, or lyophilization processes.

  The pharmaceutical composition can be provided as a salt and can be formed with hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or other protic solvents than the corresponding free base form. In other cases, a preferred formulation may be a lyophilized powder combined with a buffer prior to use, which may include any or all of the following: 1-50 mM histidine, 0.1% -2% sucrose, and 2-7% mannitol, pH range 4.5-5.5.

  After the pharmaceutical composition has been manufactured, it can be placed in a suitable container and labeled for the treatment of the indication. For administration, such labels include dosage, frequency and method.

  Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are included in an amount effective to achieve the intended purpose. Determination of an effective amount is well within the capability of those skilled in the art. For any compound, the therapeutically effective dose can be estimated initially from, for example, cell culture assays of neoplastic cells, or usually from animal models of mice, rabbits, dogs or pigs. An animal model can also be used to determine the appropriate concentration range and route of administration. Such information can be used to determine useful doses and routes for administration in humans. A therapeutically effective dose refers to that amount of active ingredient that ameliorates the symptoms or condition. Therapeutic effects and toxicity, eg LD50 (dose that 50% of the population is lethal) and ED50 (dose that 50% of the population is therapeutically effective) are measured by cell culture or standard pharmacological methods of laboratory animals. be able to. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used to formulate a dosage range for use in humans. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage will vary within this range depending upon the mode of administration used, patient sensitivity and route of administration. The exact dosage will be determined by the practitioner, based on factors related to the subject that requires treatment. Dosage amount and administration are adjusted such that sufficient levels of the active moiety are obtained, but the desired effect is maintained. Factors that may be considered include the severity of the disease state, the subject's general health, diet, time and frequency of administration, drug combination, response sensitivity, and tolerance / responsiveness to treatment. Long-acting pharmaceutical compositions can be administered every 3-4 days, every week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

  Usual dosages range from 0.1 to 100,000 micrograms, with a maximum tolerated capacity of about 1 g, and can vary depending on the route of administration. Criteria regarding specific dosages and delivery methods are obtained from literature generally available to those skilled in the art. Those skilled in the art use formulations for nucleotides that are different from the protein or its inhibitor. Similarly, delivery of polynucleotides or polypeptides is specific to a particular cell, condition, location, etc.

  Another use of the compositions of the present invention includes non-therapeutic uses, including but not limited to biomarker applications, diagnostic and research uses. One of ordinary skill in the art can use the compounds of the present invention for these purposes based on the novel discovery of mir-208-2 disclosed herein. Specific methods of research use include the reduction or increase in intracellular expression of mir-208-2 microRNA. The invention itself also reduces the expression of mir-208-2, mir-208 and / or mir-499 in cells, kits for use in diagnosing cardiovascular disorders or muscle diseases or determining their treatment strategy. Or a method of increasing and a method of decreasing or increasing mir-208-2, mir-208 and / or mir-409 activity in a cell.

Example:
Identification and characterization of miRNA-208-2.
MicroRNA expression profiling was obtained according to the following protocol:

RNA isolation Rats and mice were deeply anesthetized with isoflurane (3%, 20 L / min) and then thoracotomized to perfuse the left ventricle of the heart. 23-gauge needle with winged infusion set (SV-19BLK; Termudo, Elkton, MD) with the left ventricle connected to a hermetic pressure syringe containing wash solution (NaCl 0.9% and 250,000 U / l heparin, 38 ° C.) Punctured with. A puncture was made to provide an outlet in the right atrium, and the perfusate was injected under a precisely controlled pressure of 120 mmHg. Perfusion was continued for 2 minutes at a constant rate (20 ml). The organ was removed, snap frozen with liquid nitrogen, and stored at -80 ° C. Organs were homogenized with a Polytron homogenizer in Trizol / 100 mg tissue in the presence of 1 ml Trizol® Reagent (Life-Technologies ™, catalog number: 15596-018) according to the protocol obtained from the manufacturer. . RNA was dissolved in RNase free water and stored at -80 ° C.

Northern DNA probes mir-208 (5'-acaagctttttgctcgtcttat-3 '), mir-208-2 (5'-acaaaccttttgttcgtcttat-3'), mir-499 (5'-aaacatcactgcaagtctt-3 '), mir-206 (5' Probes for -ccacacacttccttacattcca-3 ') and U6 snRNA (5'-gccatgctaatcttctctgtatc-3') were 5'-digoxigenin labeled. All probes and synthetic miRNA sequences were obtained from Microsynth GmbH.

Northern blotting Northern blot analysis was performed using digoxigenin labeled DNA oligonucleotides. Briefly, 5 μg of total RNA from each tissue was separated on a denaturing 15% polyacrylamide / 7M urea gel (Invitrogen, catalog number: EC68855BOX) in 1 × TBE. The degraded RNA was transferred to a positively charged nylon membrane (Roche, catalog number: 1209299) for 90 minutes at 0.8 mA / cm ² in 0.5 × TBE. After UV crosslinking at 120 mJ, the membrane was washed with 2 × SSC and blocked with DIG Easy Hyb-buffer (Roche, catalog number: 11603558001) for 20 minutes. After blocking, the membrane was incubated for 60 minutes with DIG Easy Hyb-buffer containing 1 pmol / ml 5′-DIG labeled DNA oligo. The membrane was rinsed with 0.1% SDS / 2xSSC and then washed twice with 2xSSC. Subsequently, membranes were washed, blocked (Roche, Cat #: 1585762, Roche) and incubated with alkaline phosphatase conjugated anti-digoxigenin antibody (Roche, Cat #: 1093274) according to the manufacturer's protocol. Membranes were incubated in ready-to-use CDP-Star (Roche, catalog number: 2041677) and chemiluminescence was detected using ChemiDoc XRS (BioRad).

Results: miRNA expression profiling of different mouse tissues revealed that the expression of both mir-208 and mir-499 is highly enriched in the heart. Northern blot analysis of both mouse and rat tissues confirms the observed expression pattern of these miRNAs (data not shown).

  More specifically, mir-208 was highly expressed in the heart atria and ventricles, and this expression was found to be conserved in both mice and rats. On the other hand, Mir-499 is limited to the ventricular region of the heart. mir-206 is predominantly expressed in muscle and low levels of expression are detected in the heart. Based on these findings, the present inventors have shown that mir-499 (Bentwich, I., et al., Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet, 2005.) is extremely abundant in the ventricular region of the heart. And mir-208 (Lagos-Quintana, M., et al., Identification of tissue-specific microRNAs from mouse. Curr Biol, 2002. 12 (9): p. 735-9. ) And mir-206 (Sempere, LF, et al., Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol, 2004. 5 (3): p. R13 .) Heart and muscle enrichment expression profiles were confirmed.

  The mir-208 cloned from the heart (Lagos-Quintana, M., et al., New microRNAs from mouse and human. Rna, 2003. 9 (2): p. 175-9.) Is the myosin heavy chain 6 gene. Located in the intron, it is highly conserved among animals. Interestingly, mir-499 (Bentwich, et al. Supra) is located in the intron of the human myosin heavy chain 7B gene. miRNA mir-499 is highly conserved across a larger species group compared to mir-208 (zebrafish, human, chimpanzee, dog, rat, mouse and Xenopus).

  This data suggests that both gene and miRNA function are conserved, so surprisingly, a similar level of mir-499 conservation is not seen in mir-208. Interestingly, the zebrafish (Danio rerio) ventricular myosin heavy chain (vmhc) is a closely related homolog of human MYH6, whose transcription has a similar intron / exon structure.

  The alignment of the MYH6 intron containing the mir-208 sequence and the vmhc transcript revealed the presence of a class mir-208 sequence with four different nucleotides (FIG. 1). Interestingly, vmhc is more related to mammalian MYH7 family members than MYH6 members because both vmhc and MYH7 have slow contraction rates due to the slow rate of ATP hydrolysis. On the other hand, MYH6 belongs to the 'fast' isoform (Weiss, A. and L.A. Leinwand, The mammalian myosin heavy chain gene family. Annu Rev Cell Dev Biol, 1996. 12: p. 417-39). Surprisingly, the alignment of the vmhc intron containing mir-208-2 and the mammalian MYH7 intron revealed a novel mir-208-like miRNA, referred to herein as mir-208-2.

  In summary, the inventors have identified a novel miRNA sequence contained in the intron of the zebrafish gene vmhc and the mammalian MYH7 gene.

Example: Characterization of mir-208-2.
In normal mouse and rat hearts, MYH7 is expressed only during cardiac neonatal development (Lyons, GE, et al., Developmental regulation of myosin gene expression in mouse cardiac muscle. J Cell Biol, 1990. 111 (6 Pt 1): p. 2427-36.). However, MYH7 and other fetal genes are expressed again when the heart is exposed to pressure overload that results in hypertrophy. To verify the presence of mir-208-2, total RNA was isolated from mouse heart at different stages of development. RT-PCR (Figure 2) and Northern blot analysis (Figure 3) were performed to confirm gene expression and miRNA expression, respectively.

RNA Isolation Mouse hearts at different developmental stages ranging from postnatal day of birth (ED) 17-19 days were isolated, snap frozen in liquid nitrogen and stored at -80 ° C. Organs were homogenized with a Polytron homogenizer in Trizol / 100 mg tissue in the presence of 1 ml Trizol® Reagent (Life-Technologies ™, catalog number: 15596-018) according to the protocol obtained from the manufacturer. . RNA was dissolved in RNase free water and stored at -80 ° C.

Northern DNA probes mir-208 (5'-acaagctttttgctcgtcttat-3 '), mir-208-2 (5'-acaaaccttttgttcgtcttat-3'), mir-499 (5'-aaacatcactgcaagtctt-3 '), mir-206 (5' Probes for -ccacacacttccttacattcca-3 ') and U6 snRNA (5'-gccatgctaatcttctctgtatc-3') were 5'-digoxigenin labeled. All probes and synthetic miRNA sequences were obtained from Microsynth GmbH.

Northern blotting Northern blot analysis was performed using digoxigenin labeled DNA oligonucleotides. Briefly, 5 μg of total RNA from each tissue was separated on a denaturing 15% polyacrylamide / 7M urea gel (Invitrogen, catalog number: EC68855BOX) in 1 × TBE. The degraded RNA was transferred to a positively charged nylon membrane (Roche, catalog number: 1209299) for 90 minutes at 0.8 mA / cm ² in 0.5 × TBE. After UV crosslinking at 120 mJ, the membrane was washed with 2 × SSC and blocked with DIG Easy Hyb-buffer (Roche, catalog number: 11603558001) for 20 minutes. After blocking, the membrane was incubated for 60 minutes with DIG Easy Hyb-buffer containing 1 pmol / ml 5′-DIG labeled complementary DNA oligo. The membrane was rinsed with 0.1% SDS / 2xSSC and then washed twice with 2xSSC. Subsequently, membranes were washed, blocked (Roche, Cat #: 1585762, Roche) and incubated with alkaline phosphatase conjugated anti-digoxigenin antibody (Roche, Cat #: 1093274) according to the manufacturer's protocol. Membranes were incubated in ready-to-use CDP-Star (Roche, catalog number: 2041677) and chemiluminescence was detected using ChemiDoc XRS (BioRad).

Gene Expression Analysis MYH6 (Mm00440354_m1), MYH7 (Mm00600555_m1) and 18S (Hs99999901-s1) PCR primer sets from Applied Biosystems (AB) according to the manufacturer's protocol from a one-step RT-PCR Master Mix reagent (AB, catalog) No .: 4309169). All samples were measured in triplicate using 7500 FAST Real-Time PCR System (AB).

  The inventors have confirmed that MYH7 expression is restricted to the neonatal stage of mouse heart development. Two days after birth, MYH7 is no longer detected by RT-PCR. mir-208-2 is expressed before and shortly after birth (day 8), but is not substantially detected until day 14. Northern blot analysis of mir-208 correlates with MYH6 expression and is present in the neonatal and postnatal stages.

Mir-208-2 expression in adult human hearts: Unlike mice and rats, healthy human hearts express both MYH6 and MYH7. (Schiaffino, S., et al., Myosin changes in hypertrophied human atrial and ventricular myocardium. A correlated immunofluorescence and quantitative immunochemical study on serial cryosections. Eur Heart J, 1984. 5 Suppl F: p. 95-102.) Detected both MYH6 and MYH7 in autopsy and biopsy specimens of human heart using specific anti-myosin antibodies. The authors report that MYH6 is less than 5% in most normal ventricular specimens and disappeared completely due to the effects of pressure overload. On the other hand, heavy chain beta is generally not detected in the left atrial myocardium, but increased to 90% in hypertrophic atrial biopsies. Sato et al. (Sato, H., et al., [MRNA detection of beta-myosin heavy chain gene in the autopsy cases of hypertrophic cardiomyopathy]. Nippon Hoigaku Zasshi, 2000. 54 (3): p. 408-13) , MYH7 overexpression is associated with sudden cardiac death, reporting that dysregulated MYH expression contributes to cardiac pathologic dysfunction. (Garcia-Castro, M., et al., Hypertrophic cardiomyopathy: low frequency of mutations in the beta-myosin heavy chain (MYH7) and cardiac troponin T (TNNT2) genes among Spanish patients. Clin Chem, 2003. 49 (8) Perrot, A., et al., Prevalence of cardiac beta-myosin heavy chain gene mutations in patients with hypertrophic cardiomyopathy. J Mol Med, 2005. 83 (6): p. 468-77).

  The identification of a novel microRNA in the MYH7 intron established that the dysregulated expression of this miRNA is actually related to the cardiac damage caused by MYH7 itself. The present specification provides compositions and methods related to this discovery.

  All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention belongs. All publications, patents and patent applications mentioned in this specification are hereby incorporated by reference in their entirety.

Claims (24)

  An isolated nucleic acid molecule of less than 500 nucleotides, characterized in that it comprises mir-208-2 (SEQ ID NO: 7).   2. The isolated nucleic acid molecule of claim 1, wherein the length of the isolated nucleic acid molecule is less than 200 nucleotides.   2. The isolated nucleic acid molecule of claim 1, wherein the length of the isolated nucleic acid molecule is less than 100 nucleotides.   The isolated nucleic acid molecule according to any of claims 1 to 3, wherein the isolated nucleic acid molecule is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.   The isolated nucleic acid molecule of any of claims 1 to 4, wherein the isolated nucleic acid molecule consists of SEQ ID NO: 7.   An isolated nucleic acid molecule of less than 500 nucleotides, comprising an nucleic acid sequence that is complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 Nucleic acid molecule.   7. The isolated nucleic acid of claim 6 consisting of SEQ ID NO: 8.   An isolated nucleic acid molecule having a length of 8-50 nucleotides, which hybridizes with the isolated nucleic acid molecule of claim 6 under physiological conditions, preferably in a cell, of mir-208-2 (SEQ ID NO: 7). An isolated nucleic acid molecule capable of inhibiting function.   9. The isolated nucleic acid molecule of claim 8, selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 77. A) 3 'cap as desired;
b) 5 'cap;
An antisense oligodeoxynucleotide (ASO) or double-stranded oligoribonucleotide (dsRNA) comprising one or more chemical modifications selected from c) modified internucleoside linkages; or d) modified sugars or base moieties. The nucleic acid according to 8 or 9. a) 3 'cap;
b) 5 'cap;
10. A nucleic acid according to any of claims 1 to 9, comprising one or more chemical modifications selected from c) modified internucleoside linkages; or d) modified sugar or base moieties.   A nucleic acid vector comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8 and at least one vector propagation sequence.   The isolated nucleic acid molecule according to any one of claims 1 to 6, for use as a medicament.   A composition comprising the nucleic acid molecule of claim 1 or 6 in a lipid or polymer-based pharmaceutical delivery system.   11. A nucleic acid vector according to claim 10 for use as a medicament.   A cell comprising the nucleic acid vector of claim 12.   14. A method of treating muscle disease comprising administering to a subject having a muscle disease a composition comprising the isolated nucleic acid molecule of claim 13.   14. A method of treating a cardiovascular disorder comprising administering to a subject having a cardiovascular disorder a composition comprising the isolated nucleic acid molecule of claim 13.   Use of the isolated nucleic acid molecule according to any of claims 1 to 6 for the manufacture of a medicament for the treatment of muscular diseases or cardiovascular disorders.   A kit for use in the diagnosis of a cardiovascular disorder or the determination of a treatment strategy, wherein the nucleic acid molecule according to any of claims 1-9 in RNA, DNA, mixed RNA or DNA and optionally any chemistry A kit comprising a nucleic acid reagent comprising a modification.   A method for reducing or increasing mir-208-2 expression in a cell comprising administering to the cell a composition comprising an isolated nucleic acid molecule according to any of claims 1-6.   An isolated nucleic acid molecule of less than 500 nucleotides, characterized in that it comprises mir-208 (SEQ ID NO: 6), and / or mir for use in the manufacture of a medicament for the treatment of muscle disease or cardiovascular disorder An isolated nucleic acid molecule of less than 500 nucleotides characterized by comprising -499 (SEQ ID NO: 9) and / or the complementary sequence of mir-208 (SEQ ID NO: 6) or mir-499 (SEQ ID NO: 9) Use of isolated nucleic acid molecules of less than 500 nucleotides.   An isolated nucleic acid molecule of less than 500 nucleotides and / or mir-499 (sequence) for use in the treatment of a muscular disease or cardiovascular disorder comprising mir-208 (SEQ ID NO: 6) An isolated nucleic acid molecule of less than 500 nucleotides, characterized in that it comprises no. 9) and / or less than 500 comprising a complementary sequence of mir-208 (SEQ ID NO: 6) or mir-499 (SEQ ID NO: 9) Use of isolated nucleic acid molecules of nucleotides.   24. A method of treating a patient comprising the use of a medicament according to claim 19, 22 or 23.

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